The bacterial stringent response system that controls transitions between growth and non-growth states in E. coli has not been evaluated with respect to its role in animal colonization. The stringent response to nutrient limitation involves the rapid accumulation of a unique nucleotide, guanosine 3', 5'-bispyrophosphate (ppGpp), which causes extensive reprogramming of gene expression during growth transitions. Synthesis and degradation of ppGpp is dynamic, allowing the cell to rapidly adapt to stress and then rapidly resume growth when conditions allow. The proposed research plan will determine whether intestinal colonization is dependent on maximal growth rate of the bacteria in the intestine or the ability of the bacteria to respond to nutrient limitation and adapt to the intestinal environment. Specifically, we will address four questions with regard to nutrient availability in the mouse intestine and the adaptation of E. coli to the intestinal environment. (1) Does E. coli lead a feast and famine existence in the mouse intestine? We have identified ppGpp-dependent genes to use as molecular beacons of the growth status of the cell, which will allow us to ask whether or not E. coli experiences discontinuous or continuous nutrient availability in the intestine. Gene fusions to fluorescent protein reporters will be used to visualize with confocal microscopy the growth status of individual bacterial cells within the mucus layer of infected mouse intestines. Alternatively we will use in situ hybridization to measure expression of these growth regulated genes in single bacterial cells. These experiments will reveal the nature of the intestinal environment as it is perceived by colonized bacteria. (2) What is the minimum growth rate required to sustain colonization? Experimentally, we will address this question by infecting mice with a series of strains that have been genetically manipulated to fix their ppGpp levels independently of environmental signals and hence have fixed growth rates. We will determine the relative fitness of these strains for colonization and measure their in vivo growth rates. (3) Do faster growing strains out-compete strains with slower growth? The strains with fixed growth rates will be competed against each other. (4) Is the ability to adjust ppGpp levels (i.e., growth rate) more important than sustaining the maximal logarithmic growth rate? The fixed ppGpp strains will be competed with the wild type, which can adjust its growth rate normally. These experiments, to be conducted in the model organism E. coli K-12, should lead to novel insights into the physiology of colonized bacteria. Because colonization is the first step in infection and disease, this work will be extended to E. coli O157:H7 in the future to search for common themes in commensalism and pathogenesis. The answers to these questions will lead to a better understanding of gastrointestinal health and potentially to novel strategies for combating gastrointestinal infections. PUBLIC HEALTH RELEVANCE: This proposal is based on the hypothesis that E. coli must appropriately control its growth rate to colonize and achieve high populations in the intestine. In testing this hypothesis, we will determine whether animal colonization is dependent on maximal growth rate of the bacteria in the intestine or the ability of the bacteria to respond to nutrient limitation and adapt by adjusting their growth rate. The answers to these questions will provide new information about the physiology of intestinal bacteria within their host and a better understanding of gastrointestinal health, which will lead to novel strategies for combating gastrointestinal infections.